Methods for selectively occluding blood supplies to neoplasias

ABSTRACT

Disclosed are methods of selectively reducing the blood supply to a neoplastic region, such as a tumor region, thereby selectively causing necrosis of the neoplastic tissue without substantial necrosis of adjoining tissues. In particular, methods are disclosed of selectively reducing the blood supply to a neoplastic region, such as a tumor region, by causing selectively occlusion of blood vessels feeding the neoplastic region. The invention also provides methods of selectively causing anti-angiogenic action in a neoplastic region, such as a tumor region, with the result that new blood vessels are not formed to sustain the neoplasia. The methods employ intra-arterial injection of polyunsaturated fatty acids, preferably in the form of salts, preferably with a lymphographic agent, and optionally with an anti-cancer drug, and/or a cytokine. The invention also provides solutions of PUFAs, or salts of PUFAs, in combination with a lymphographic agent.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/946,129, filed Sep. 4, 2001, which is a continuation-in-partof U.S. patent application Ser. No. 09/392,953, filed Sep. 9, 1999, thedisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods for selectively occluding bloodvessels which supply neoplastic tissue, including tumors.

[0004] 2. Description of the Related Art

[0005] The polyunsaturated fatty acids (PUFAs) are fatty acids having atleast two carbon-to-carbon double bonds in a hydrophobic hydrocarbonchain which typically includes X-Y carbon atoms and terminates in acarboxylic acid group. The PUFAs are classified in accordance with ashort hand nomenclature which designates the number of carbon atomspresent (chain length), the number of double bonds in the chain and theposition of double bonds nearest to the terminal methyl group. Thenotation “a:b” is used to denote the chain length and number of doublebonds, and the notation “n-x” is used to describe the position of thedouble bond nearest to the methyl group. There are 4 independentfamilies of PUFAs, depending on the parent fatty acid from which theyare synthesized. They are:

[0006] (1) The “n-3” series derived from alpha-linolenic acid (ALA,18:3, n-3).

[0007] (2) The “n-6” series derived from linoleic acid (LA, 18:2, n-6).

[0008] (3) The “n-9” series derived from oleic acid (OA, 18:1, n-9).

[0009] (4) The “n-7” series derived from palmitoleic acid (PA, 16:1,n-7).

[0010] The parent fatty acids of the n-3 and n-6 series can not besynthesized by the mammals, and hence they are often referred to as“essential fatty acids” (EFAs). Because these compounds are necessaryfor normal health but cannot be synthesized by the human body, they mustbe obtained through the diet.

[0011] It is believed that both LA and ALA are metabolized by the sameset of enzymes. LA is converted to gamma-linolenic acid (GLA, 18:3, n-6)by the action of the enzyme delta-6-desaturase (d-6-d), and GLA iselongated to form di-homo-GLA (DGLA, 20:3, n-6), the precursor of the 1series of prostaglandins. The reaction catalyzed by d-6-d is the ratelimiting step in the metabolism of EFAs. DGLA can also be converted toarachidonic acid (AA, 20:4, n-6)) by the action of the enzymedelta-5-desaturase (d-5-d). AA forms the precursor of 2 series ofprostaglandins, thromboxanes and the 4 series leukotrienes. ALA isconverted to eicosapentaenoic acid (EPA, 20:5, n-3) by d-6-d and d-5-d.EPA forms the precursor of the 3 series of prostaglandins and the 5series of leukotrienes. Conjugated linoleic acid (CLA; 18:2) is a groupof isomers (mainly 9-cis, 11-trans and 10-trans, 12-cis) of linoleicacid. CLA is the product of rumen fermentation and can be found in themilk and muscle of ruminants (see, e.g., Brodie et al. (1999), J. Nutr.129: 602-6; Visonneau et al. (1997), Anticancer Res. 17: 969-73. LA,GLA, DGLA, AA, ALA, EPA, docosahexaenoic acid (DHA, 22:6, n-3) and CLAare all PUFAs, but only LA and ALA are EFAs.

[0012] Under some well defined culture conditions GLA, AA, EPA and DHAshowed a marked differential cytotoxic effect against tumor cells withlittle or no significant action on normal cells (Leary et al. (1987), S.Afr. Med. J. 62:681-683; Begin et al. (1985), Prostaglandins Leukot.Med. 19:177-186; Das (1999), Nutrition 15:239-241; Das (1991), CancerLett. 56:235-243; Das (1990), Nutrition 6:429-434; Seigel et al. (1987),J. Natl. Cancer Inst. 78:271-277; Sangeetha and Das (1992), Cancer Lett.63:189-198; Begin et al. (1986), J. Natl. Cancer Inst. 77:1053-1062; Das(1992), Asia Pacific J. Pharmacol. 7:305-327). In mixed cultureexperiments, in which both normal and tumor cells were grown together,GLA showed more selective tumoricidal action compared to AA and EPA(Begin et al. (1986), Prog. Lipid Res. 25:573-576). In addition, directintra-tumoral administration of GLA can regress human gliomas withoutsignificant side-effects (Naidu et al. (1992), Prostaglandins Leukot.Essen. Fatty Acids 45: 181-184; Das et al. (1995), Cancer Lett.94:147-155).

[0013] Thus, it is known in the art that certain polyunsaturated fattyacids (PUFAs) have cytotoxic properties towards tumor cells in vitro,and that PUFAs provide the substrates for the generation of lipidperoxidation products which have an inhibitory action on cellproliferation. In addition, tumor cells are known to have low d-6-dactivity, which is necessary for the desaturation of LA and ALA to theirrespective products. Moreover, it has been shown that hepatocarcinogens,diethylnitrosamine (DEN) and 2-acetylamino-fluorine (2-AAF), cansuppress the activity of d-6-d and d-5-d resulting in low levels of GLAand AA, EPA and DHA in the tumor cells.

SUMMARY OF THE INVENTION

[0014] In one aspect, the present invention provides methods ofselectively interrupting the blood supply to a neoplastic region, suchas a tumor region, causing necrosis of the neoplastic tissue withoutsubstantial necrosis of adjoining tissues. The invention also providesmethods of selectively causing anti-angiogenic action in a neoplasticregion, such as a tumor region, with the result that new blood vesselsand collaterals are not formed to sustain the neoplasia.

[0015] In particular, the invention provides methods for selectivelyreducing blood supply to at least a portion of a neoplastic region, inwhich (a) a proximal artery which carries blood to at least a portion ofsaid region is located and (b) a therapeutically effective amount of asolution of at least one PUFA is intra-arterially injected into theartery, thereby selectively reducing the blood supply. In preferredembodiments, the amount of the solution is sufficient to cause occlusionof the artery in a period of less than one minute. In preferredembodiments, the therapeutically effective amount is between 0.5 mg and50 gm, most preferably between 250 mg and 5 gm.

[0016] In some embodiments of the invention, in addition to the PUFA, alymphographic agent is intra-arterially injected to visualize theproximal artery and blood supply to the neoplastic region. Thelymphographic agent may be combined with the PUFA solution and they maybe injected together. The progress of the lymphographic agent throughthe proximal artery and neoplastic region can be observed to determinewhen the blood supply is effectively reduced and when injection of thePUFA solution can be stopped. In some embodiments, the lymphographicagent is covalently conjugated to the PUFA.

[0017] In some embodiments, the PUFA is an EFA. In certain preferredembodiments, the EFA is selected from linoleic acid, gamma-linolenicacid, arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid,di-homo-gamma-linolenic acid, alpha-linolenic acid, linoleic acid, andconjugated linoleic acid.

[0018] In preferred embodiments, the PUFA is administered in the form offree acid or a salt, such as a lithium salt, a sodium salt, magnesiumsalt, a manganese salt, an iron salt, a copper salt or an iodide salt.In some preferred embodiments, the PUFA is in the form of a fatty acidderivative, such as a glyceride, ester, ether, amide, or phospholipid,or an alkylated, alkoxylated, halogenated, sulfonated, or phosphorylatedform of the fatty acid.

[0019] In some embodiments of the inventions, the neoplastic tissue is atumor. In particular, the neoplastic tissue may be a glioma, hepatoma,lung cancer, colon cancer, breast cancer, ovarian cancer, kidney cancer,skin cancer, Kaposi's sarcoma, esophageal cancer, stomach cancer,leukemia, or lymphoma. In other embodiments, the neoplastic tissue mayresult from a non-cancerous cell proliferative disorder.

[0020] In some embodiments of the invention, in addition to the PUFA, atherapeutically effective amount of a compound selected from tumornecrosis factor, anticancer drugs, lymphokines, and specific polyclonalor monoclonal antibodies is intra-arterially injected. In preferredembodiments, the lymphokine is alpha interferon or gamma interferon.

[0021] In some embodiments, the PUFA is covalently conjugated with apharmaceutical agent chosen from TNF, alpha-interferon,gamma-interferon, an antibody, vincristine, adriamycin, doxorubicin,cyclophosphamide, cis-platinum, L-asparaginase, procarbazine,camptothecin, taxol or busulfan.

[0022] In another aspect, the invention provides pharmaceuticalcompositions of a PUFA, or salt of PUFA, in combination with alymphographic agent or anti-neoplastic agent.

DETAILED DESCRIPTION

[0023] The patent, scientific and medical publications referred toherein establish knowledge that was available to those of ordinary skillin the art at the time the invention was made. The entire disclosures ofthe issued U.S. patents, published and pending patent applications, andother references cited herein are hereby incorporated by reference.

[0024] Definitions.

[0025] In order to more clearly and concisely describe the subjectmatter which is the invention, the following definitions are providedfor certain terms which are used in the specification and appendedclaims.

[0026] As used herein, the term “neoplastic” means characterized byabnormal tissue that shows partial or complete lack of structuralorganization and functional coordination with normal tissue, and usuallyforms a distinct mass which may be either benign or malignant. As usedherein, “neoplastic” tissue need not exhibit cellular proliferation thatis more rapid than normal tissue (e.g., a tumor which has ceased to growor which is in remission). Neoplastic tissue need not be cancerous(e.g., uterine fibroids, adenomatous polyps of the intestines, adenomasin the lungs or other organs).

[0027] As used herein, a “neoplastic region” means an essentiallycontiguous region of tissue containing neoplastic tissue. A neoplasticregion is the smallest volume of tissue that includes the contiguousneoplastic tissue, but may also include normal tissue. Contiguousneoplastic tissues are neoplastic tissues separated by distances of lessthan one centimeter, and do not include distant metastases (which defineseparate neoplastic regions). Although not all neoplastic regions aretumors, the terms “neoplastic region” and “tumor” will often be usedinterchangeably herein, and the term “tumorfeeding vessel” should beunderstood to include an artery feeding any type of neoplastic region.

[0028] As used herein, the term “polyunsaturated fatty acid” and theabbreviation “PUFA” mean any acid derived from fats by hydrolysis, orany long-chain (at least 12 carbons) organic acid, having at least twocarbon-to-carbon double bonds. Examples of PUFAs include but are notlimited to linoleic acid, linolenic acid and arachidonic acid.

[0029] As used herein, the term “PUFA salt” means an ionic association,in solid or in solution, of a anionic form of a PUFA with a cation of asmall organic group (e.g., ammonium) or a small inorganic group (e.g.,an alkali metal). Preferred salts are those between a PUFA and an alkalimetal (e.g., lithium, sodium, potassium), an alkali earth metal (e.g.,magnesium, calcium) or a multivalent transition metal (e.g., manganese,iron, copper, aluminum, zinc, chromium, cobalt, nickel).

[0030] As used herein the term “lymphographic agent” means any of theclass of compounds which are used, or may be used, to visualizelymphatics and lymph nodes, as well as veins and arteries, following anintravenous or intra-arterial injection. Lymphographic agents aretypically vegetable oils (e.g., poppy seed oil) which are iodized (e.g.,approximately 30-45% by weight), and which may be further derivatized(e.g., ethyl esterification). Examples include the iodized fatty acidsof poppy seed oil (commercially available as LIPIODOL ULTRA FLUIDE® fromLaboratoire Guerbet, Paris, France), the ethiodized fatty acids of poppyseed oil (commercially available as ETHIODOL® from Savage Laboratories,Melville, N.Y.) and iophendylate (PANTOPAQUE® from Kodak). See, Hom etal. (1957), J. Am. Pharm. Assoc. Sci. Ed. 46:254; Paxton et al. (1975),Brit. Med. J. 1:120. As used herein, the term “lymphographic agent”means any agent which is useful for non-invasively visualizing bloodvessels including, without limitation, radiography, CAT scans, MRIscans, ultrasound imaging, and the like.

[0031] As used herein, the term “angiogram” means any method ofnoninvasively visualizing a blood vessel or lymphatic including, withoutlimitation, radiography, CAT scans, MRI scans, ultrasound imaging, andthe like.

[0032] As used herein, the term “proximal” is a relative term whichdescribes the location of an artery with respect to a neoplastic regionand a site of intra-arterial injection of a PUFA salt of the invention.An artery is proximal to a neoplastic region if it is upstream of theneoplastic region with respect to blood flow and downstream (or distal)of the site of injection with respect to blood flow. A proximal arteryshould also be physically close to the neoplastic region such that asubstantial portion (e.g., at least 10%, preferably 25%, most preferablygreater than 50%) of the volume of a solution injected into the arterywould normally pass into arteries, arterioles and capillary beds withinthe neoplastic region.

[0033] General Considerations

[0034] The present invention is dependent, in part, upon the discoveryof the novel and highly beneficial action of PUFAs, and especiallycertain PUFA salts, to induce the selective occlusion of blood vesselsfeeding neoplastic regions, including tumors. This effect isparticularly observed when the PUFA is administered in combination witha lymphographic agent comprising iodized fatty acids.

[0035] Without being bound to any particular theory of the invention, itis believed that the selective occlusion of the tumor-feeding vessels isnot due to embolism or other forms of physical blockage. This conclusionfollows from observations in several patients that normal blood vessels,which were sometimes smaller in diameter than tumor-feeding vessels,which were located proximal to the tumor-feeding vessels, and which werecloser to the tip of the catheter and the site of injection, were notoccluded. If the occlusion were due to embolization, all blood vessels,especially those that were smaller in diameter compared to thetumor-feeding vessels, would be expected to be occluded first. Becausethe site of injection of the PUFA, as determined by angiographic imagingof the tip of the catheter in several patients, was slightly upstreamfrom the origin of the main tumor-feeding vessels, it is evident thatthe occlusion of the tumor-feeding vessels is not due to directinjection of the PUFA only into those vessels. Rather, the ability ofPUFA to selectively occlude the tumor-feeding vessels but not normalarterial vessels was seen in several patients.

[0036] Moreover, in several other patients, a PUFA was injected intonormal arteries including the celiac, subclavian and popliteal arteries.During the course of these procedures, no spasms or occlusions (eventemporary) of these blood vessels were observed. On the other hand, thePUFA occluded all types of tumor-feeding vessels, irrespective of theirsize, almost instantaneously. Without being bound to any particulartheory of the invention, this rapid action of the PUFA suggests that anintense vasospasm was induced (directly or indirectly) in thetumor-feeding vessels but not in the normal blood vessels and that,following such a vasospasm, thrombosis may have led to permanentocclusion of the blood vessel.

[0037] Finally, without being bound to any particular theory of theinvention, it is believed that there is an interaction between the PUFAand lymphographic agents of the invention which may account for theeffectiveness of the treatment. Thus, lymphographic agents comprisingiodized fatty acids, and particularly the iodized fatty acids ofvegetable oils, are believed to synergistically interact with the PUFAsto produce a therapeutic effect which is qualitatively different thanthe effect of either the PUFA or the lymphographic agent alone.

[0038] There are several advantages of PUFA treatments of the invention.As shown below, a single injection (or at most two or three injectionsat separate times, if the neoplastic region is large) is adequate toproduce almost permanent occlusion of the tumor-feeding vessels. ThePUFAs and their salts are non-antigenic, are known to be relatively safein the dosages employed, and are stable. The dosage of PUFA needed toocclude the tumor-feeding vessels in a given patient is self-evidentduring administration: As the PUFA solution is being injected, and asthe tumor-feeding vessels are being occluded, resistance to furtherinjection will be felt, at which point the injection can be stopped.

[0039] The invention in one aspect provides methods of inhibiting bloodsupply to a neoplastic region, comprising the steps of (a) locating anartery which carries major blood supply to the neoplastic region andwhich is proximal to the neoplastic region; and (b) intra-arteriallyinjecting into the located artery a solution of at least one PUFA chosenfrom LA, GLA, DGLA, AA, ALA, EPA, DHA and CLA. In preferred embodiments,the PUFA is administered in combination with a lymphographic agent.

[0040] The invention in another aspect provides methods for treatingneoplasias and for facilitating the visualization of remission of aneoplasia which is responsive to treatment, comprising the steps of (a)locating an artery proximal to the neoplastic region which carries amajor portion of blood supply to the neoplastic region and which isadjacent to the neoplastic region; (b) obtaining an initial radiographicimage of the region; (c) injecting into the artery a mixture of (i) alymphographic agent, and (ii) a solution of at least one PUFA chosenfrom LA, GLA, DGLA, AA, ALA, EPA, DHA and CLA; and (d) obtaining secondand, optionally, subsequent radiographic images of the neoplastic regionafter predetermined lapses of time; and comparing the initialradiographic images with the second and/or subsequent radiographicimages to assess the extent of remission of the neoplasia.

[0041] The invention in another aspect provides methods of causingnecrosis in a neoplastic region (e.g., a cancerous tumor) by inhibitingblood supply to the neoplastic region, comprising the steps of (a)locating an artery proximal to the neoplastic region which carries majorblood supply to the neoplastic region; (b) injecting into the locatedartery a mixture of (i) a lymphographic agent, and (ii) a solution of atleast one PUFA chosen from LA,GLA, DGLA, AA, ALA, EPA, DHA and CLA; (c)waiting for a predetermined time period and assessing a degree ofnecrosis in the neoplastic region; and (d) repeating the treatment ifnecessary to increase the necrosis.

[0042] In yet another aspect, the invention provides methods of treatingmammalian cell proliferative disorders using a solution of a PUFA, orcombinations of PUFAs, administered intra-arterially. The methods are asdescribed above with respect to neoplastic regions.

[0043] In each of the foregoing embodiments, the PUFA is preferably inthe form of a salt, most preferably in the form of a lithium salt, andis preferably administered in combination with a lymphographic agent.The lymphographic agent is preferably an iodized fatty acid derived froma vegetable oil.

[0044] Although the invention is described primarily as it relates tohumans, it is envisaged that the methods of the invention are equallyapplicable to other mammals, including large domesticated mammals (e.g.,race horses, breeding cattle) and smaller domesticated animals (e.g.,house pets).

[0045] Choice of PUFA

[0046] The present invention employs PUFAs, preferably in the form ofsalts, to selectively occlude arteries which provide blood supply toregions of neoplastic tissue. Preferred PUFAs include, but are notlimited to, GLA, AA, DHA, EPA, DGLA, ALA, LA and CLA. Other preferredPUFAs include derivatives of the aforementioned PUFAs, includingglycerides, esters, ethers, amides, or phospholipids, or alkylated,alkoxylated, halogenated, sulfonated, or phosphorylated forms of thefatty acid. In most preferred embodiments, the PUFA is GLA, AA or DHA.

[0047] The PUFA is preferably administered in the form of a saltsolution. Suitable salts include salts of a PUFA with a cation of asmall organic group (e.g., ammonium) or a small inorganic group (e.g.,an alkali metal or alkali earth metal). Preferred referred salts arethose between a PUFA and an alkali metal (e.g., lithium, sodium,potassium), an alkali earth metal (e.g., magnesium, calcium) or amultivalent metal (e.g., manganese, iron, copper, aluminum, zinc,chromium, cobalt, nickel). Most preferred are salts of lithium, sodium,magnesium, manganese, iron, copper, and iodides. Combinations of saltsmay also be employed.

[0048] When the PUFAs or PUFA salts are administered in combination withan oily lymphographic agent or other agents, the solution may be formedinto an emulsion.

[0049] Lymphographic Agents

[0050] In order to visualize lymphatic vessels, lymph nodes, arteriesand veins, lymphographic agents are frequently employed. In the contextof the present invention, these agents may aid in both the placement ofa syringe or catheter in a proximal artery for intra-arterial injectionof a PUFA solution, and may also aid in the visualization of theresulting selective occlusion of tumor-feeding vessels. In addition,such agents may be used in follow-up angiograms to determine whetherocclusion has been successful or complete, and to determine whetheradditional treatments may be necessary. Finally, it is believed thatsuch agents have a synergistic or potentiating effect in combinationwith the PUFA solutions of the invention, and thereby serve asadditional or ancillary active ingredients in the treatments of theinvention.

[0051] The lymphographic agents can be any of the class of compounds,recognized by those of skill in the art of diagnostic imaging, which areused, or which may be used, to visualize lymphatics and lymph nodes, aswell as veins and arteries, by radiography following an intra-lumenalinjection. Lymphographic agents are typically vegetable oils (e.g.,poppy seed oil) which are iodized (e.g., approximately 30-45% byweight), and which may be further derivatized (e.g., ethylesterification). Examples include the iodized fatty acids of poppy seedoil (commercially available as LIPIODOL ULTRA FLUIDE® from LaboratoireGuerbet, Paris, France), the ethiodized fatty acids of poppy seed oil(commercially available as ETHIODOL® from Savage Laboratories, Melville,N.Y.) and iophendylate (PANTOPAQUE® from Kodak). See, Hom et al. (1957),J. Am. Pharm. Assoc. Sci. Ed. 46:254; Paxton et al. (1975), Brit. Med.J. 1:120.

[0052] The lymphographic agents of the invention may be mixed with thePUFA solutions described above, either to form a new solution or to forman emulsion, or they may be chemically conjugated to the PUFAs of theinvention via standard chemistries. Preferably the lymphographic agentis an iodized lymphographic oil, such as an iodized poppy seed oil.Preferably the PUFA solution is mixed with such a lymphographic agent ina ratio of at least about 2:1, or about 1:1, or about 1:1.5, or about1:2, or about 1:3 (volume/volume). Most preferably the ratio is between1:1.5 and 1:3 (volume/volume). The preferred lymphographic agent isLIPIODOL ULTRA FLUIDE® (Laboratoire Guerbet, Paris, France). Thislymphographic agent may be safely administered to a typical patient inan amount of about 10 mL/m², but the attending physician should considerall relevant medical factors in determining the appropriate dosage forany specific patient.

[0053] Thus, in another aspect, the invention provides pharmaceuticalcompositions comprising a PUFA, or a PUFA salt, and a lymphographicagent in solution, or in an emulsion. The PUFA and lymphographic agentmay be separate chemical moieties combined in the solution or emulsion,or they may be covalently conjugated. The preferred lymphographic agentsand ratios for such a product are as disclosed above. Preferably thefinal concentration of the PUFA in such a product is at least 5%,preferably at least 20%, and most preferably about 25-50%.

[0054] Methods of Administration

[0055] The PUFA solutions of the present invention are preferablyadministered intra-arterially to an artery which is proximal to theneoplastic region to be treated. The approximate location of theneoplastic region must first be identified by any of the methods knownin the art. For example, X-rays, Computerized Axial Tomography (CAT)scans, Magnetic Resonance Imaging (MRI) scans, palpation or directvisual inspection may be used to identify a neoplastic region. Suchmethods may optionally employ contrast agents, including lymphographicagents or agents specifically targeted to neoplastic tissues (e.g.,radioisotope-labeled antibodies against tumor-associated antigens). Oncethe neoplastic region is identified, an artery which feeds the region(i.e., which is upstream with respect to blood flow to the region) isidentified. The intra-arterial injection site is preferably chosen to beclose or proximal to the neoplastic region to increase the portion ofthe dosage which reaches that region, but is also preferably chosensufficiently far upstream from that region such that all or most of theneoplastic region receives a portion of the injected dosage.

[0056] Thus, as one progresses along an artery which feeds a neoplasticregion, the artery will branch into smaller and smaller arteries andfinally arterioles. At some distant point upstream from the neoplasticregion, the artery will feed not only the neoplastic region but alsolarge regions of normal tissue. As the chosen injection site is movedalong the artery toward the neoplastic region, the percentage of bloodcarried by the artery which feeds normal tissue will decrease. Byproceeding along the artery toward the neoplastic region, therefore, onecan increase the portion of the dosage which reaches the neoplasticregion. However, as the injection site proceeds along the artery towardthe neoplastic region, one may also bypass branches of the artery whichfeed the neoplastic region and, therefore, fail to cause occlusion ofarteries supplying a part of the neoplastic region. One of ordinaryskill in the art may balance these considerations, as well as otherconsiderations (e.g., accessibility of an artery for catheterization),in choosing a site for injection. Thus, the term “proximal” is arelative term which describes the location of an artery with respect toa neoplastic region and a site of intra-arterial injection of a PUFA ofthe invention. Preferably, a proximal artery should also be physicallyclose to the neoplastic region such that a substantial portion (e.g., atleast 10%, preferably 25%, and most preferably greater than 50%) of thevolume of a solution injected into the artery would normally pass intoarteries, arterioles and capillary beds within the neoplastic region.Thus, the hepatic artery might be considered proximal to a neoplasticregion in the liver, but the descending aorta would not.

[0057] In order to administer a PUFA solution to a proximal artery, theartery is identified as described above, the site of injection ischosen, and the PUFA solution is administered by injection through asyringe or catheter as appropriate to the location. As necessary, thesyringe or catheter may be guided to the site of injection byradiological guidance (e.g., X-rays), CAT guidance, MRI guidance,endoscopic guidance, or stereotaxic guidance. In the case of a catheter,the catheter can be inserted into the body at a site quite distant fromthe proximal artery, and then be guided to the proximal artery. Forexample, the femoral, brachial and carotid arteries may provideconvenient entry points for a catheter which is then routed to aproximal artery elsewhere in the body. In addition, contrast agents maybe added to the injected solution to aid in placement of the syringe orcatheter, or to aid in visualization of the occlusion of thetumor-feeding vessels.

[0058] Appropriate dosages of the PUFA solutions of the invention willdepend primarily on the diameter of the proximal artery at the site ofinjection and the number and size of the arteries and/or arteriolesbranching therefrom. Preferred dosages range from approximately 0.5 mgfor the smallest proximal arteries to 50 gm for very large proximalarteries feeding large neoplastic regions. More typically, dosages ofapproximately 250 mg to 5 gm are preferred and, as shown in the examplesbelow, dosages of 500 mg to 750 mg are effective for several differenttypes of tumors. However, in most preferred embodiments of the methodsof the invention, the PUFA solution is administered in combination witha lymphographic agent, the administration is observed by angiogram, andadministration continues until the tumor-feeding vessels are at leastpartially occluded as indicated by the angiogram. Alternatively oradditionally, administration may be continued until a significantincrease in resistance to the injection develops, indicating thetumor-feeding vessels distal to the site of injection have been at leastpartially occluded.

[0059] Other Agents

[0060] The PUFA solutions of the invention may be administered alone, orin combination with other pharmaceutical agents known in the art for thetreatment of neoplasias. Thus, for example, the PUFA solutions may beco-administered with known anti-cancer drugs, including vincristine,adriamycin, doxorubicin, cyclophosphamide, cisplatinum, L-asparaginase,procarbazine, camptothecin, taxol and busulfan. Alternatively, the PUFAsolutions may be co-administered with known lymphokines such as tumornecrosis factor (TNF) and/or an interferon (e.g., alpha interferon orgamma interferon) or specific polyclonal or monoclonal antibodies.

[0061] Administration of these agents in combination with a PUFAsolution, or a PUFA and lymphographic agent solution, may also show asynergistic or potentiating effect.

[0062] Thus, in another aspect, the invention provides pharmaceuticalcompositions comprising a PUFA, or a PUFA salt, and a pharmaceuticalagent known in the art for the treatment of neoplasias, either insolution, or in an emulsion. The PUFA and other pharmaceutical agent maybe separate chemical moieties combined in the solution or emulsion, orthey may be covalently conjugated. The preferred pharmaceutical agentsare as disclosed above. Preferably the final concentration of the PUFAin such a product is at least 5%, preferably at least 15%, and mostpreferably at least 25%. The product may contain substantially morePUFA, up to 100% without any significant side-effects.

[0063] The following examples illustrate some preferred modes ofpracticing the present invention, but are not intended to limit thescope of the claimed invention. Alternative materials and methods may beutilized to obtain similar results.

EXAMPLES

[0064] Patients

[0065] Studies were conducted in 5 human patients with stage 4neoplastic disease. Two of the patients had primary hepatoma (patients 1and 2), two had giant cell tumor of the bone (patients 3 and 4) and onehad renal cell carcinoma (patient 5).

[0066] Materials

[0067] The lithium salt of GLA was obtained from Scotia Pharmaceuticals,U.K. The PUFA salt was dissolved in sterile saline, sterile phosphatebuffered saline (PBS, pH 7.4) or dilute ethanol in saline (finalconcentration <0.02% ethanol). The final concentration of PUFA in thesesolutions was approximately 25%. The PUFA solution was mixed with aniodized lymphographic oil (LIPIODOL ULTRA FLUIDE®, Laboratoire Guerbet,Paris, France), in a ratio of between 1:1.5 and 1:3 (volume/volume). Insome cases, the PUFA was modified by covalent conjugation (e.g., amidebond) to the iodized lymphographic oil. In other cases the PUFA salt wasunmodified, and was diluted directly into the lymphographic agentwithout conjugation. The PUFA and lymphographic oil mixture was preparedunder sterile conditions immediately prior to use.

[0068] Methods of Administration

[0069] Patients were admitted into the hospital for the study. Aproximal artery supplying a major portion of the blood supply to thetumor was identified. Catheterization of the major artery from which theproximal artery arises was performed under local anaesthesia. Inpatients 1 and 2 (with hepatomas), the tip of the catheter waspositioned in the right hepatic artery via the right femoral artery. Inpatient 3 (with giant cell tumor of the right lower end of the femur),the tip of the catheter was positioned in the right femoral/poplitealartery. In patient 4 (with giant cell tumor of the left scapula), thetip of the catheter was positioned in the left subclavian/axillaryarteries. In patient 5 (with left kidney tumor), the tip of the catheterwas positioned in the left renal artery. Conjugated PUFA salt wasprepared fresh, just prior to injection. Radiographic and CT scanexaminations were performed immediately after the injection and atperiodic intervals in all the five patients.

[0070] In order to determine how the arterial supply to the tumor tissuewas influenced by the injection of PUFA salt, angiography was performedand recorded during and immediately after the procedure, and at periodicintervals thereafter.

[0071] Patients 1 and 2 with hepatoma were administered total doses of1.6 gm and 0.75 gm respectively (the dose refers to the amount ofLi-GLA) of PUFA salt into the right hepatic artery through the rightfemoral route. Patient 3, with giant cell tumor of the lower end of theright femur, underwent right femoral artery catheterization and the tipof the catheter was positioned in the popliteal artery to deliver 500 mgof PUFA salt. Patient 4, who had giant cell tumor of the left scapula,received PUFA salt by selective cannulation of the left subclavianartery, through the femoral route, from which the tumor-feeding vesselwas arising and was given 660 mg of PUFA salt. Patient 5, with renaltumor, received 750 mg of PUFA salt through the right femoral route intothe left renal artery from which the tumor-feeding vessels were arising.In all the patients, the administration of PUFA salt was done as swiftlyas possible. During the administration of PUFA salt, the vital signs ofeach patient were monitored.

[0072] Summary Results

[0073] All 5 patients tolerated the treatment well and no significantside-effects due to the therapy were noted. The only side-effect was acomplaint of a mild feeling of warmth followed by pain at the site ofthe tumor during and immediately after the injection of the PUFA salt.This was presumably due to the perfusion of the tumor with the PUFAsalt, and ischaemia resulting from occlusion of the tumor-feedingvessels. In general, the pain was not severe and was ameliorated by theadministration of non-steroidal anti-inflammatory drugs (NSAIDs), suchas ibuprofen or buscopan. All the biochemical tests performed after theadministration of the PUFA salt were found to be normal.

[0074] The most significant and surprising observation of these studieswas the total occlusion of the tumor-feeding vessels following PUFA saltinjection. This was a consistent observation in all the 5 patients. Thisselective occlusion of the tumor-feeding vessels was seen during thecourse of injecting the PUFA salt in the patients with giant cell tumorsof the bone and the patient with renal cell carcinoma. In the case ofthe patients with hepatomas, the occlusion of the tumor-feeding vesselswas noticed over a period of time. In patient 1 the occlusion of thetumor-feeding vessels was observed 10 days after the first injection ofPUFA salt, and six days after a second injection, but presumablyoccurred during or shortly after the second injection. No occlusion wasseen in the normal vasculature feeding non-neoplastic tissues. The timelag for the occlusion of the tumor-feeding vessels observed in patient 1with hepatoma suggests that the occlusion of the tumor-feeding vesselsis not due to an embolization process. Moreover, normal blood vessels,which were much smaller in diameter compared to the tumor-feedingvessels, were not occluded when exposed to PUFA salt, further suggestingthat embolization was not the mechanism. On the other hand, in patient2, also with hepatoma, the occlusion of the tumor-feeding vessels wasseen essentially simultaneously with the injection of the PUFA salt. Theremarkably selective occlusion of only the tumor-feeding vessels wasclear from pre- and post-injection angiograms of patient 5 with therenal cancer. These angiograms showed that the three main tumor-feedingvessels arising from the main renal artery were completely occludedwhereas the fourth and the fifth branches arising from the main stem ofthe left renal artery, which were feeding the normal lower pole of thekidney, were not occluded. This was despite the fact that all the fivevessels were arising from the same renal artery, downstream from thepoint of injection. If the occlusion of the vessels were the result ofembolization, it is expected that the lower most and narrower branchesshould have been occluded first and later the other vessels.

[0075] The pre and post-injection angiograms of all the 5 patientsindicated that PUFA salt can selectively occlude tumor-feeding vessels.In order to know the duration of this selective occlusion of thetumor-feeding vessels, angiograms were repeated at periodic intervals.It was noted that even 28 days after PUFA salt injection, notumor-feeding vessels could be seen in patient 1 with hepatoma. In thepatient with giant cell tumor of the lower end of the right femur(patient 3), a repeat angiogram performed after 10 days following PUFAsalt injection also did not show any tumor-feeding vessels. Radiographsof the right knee of this patient taken 10 days after the injectionclearly showed the presence of the contrast material, which is due tothe presence of conjugated PUFA salt in the tumor tissue. In the patientwith giant cell tumor of the left scapula (patient 4), a follow upangiogram performed seven and a half years after the injection of PUFAsalt clearly showed that the original tumor-feeding vessel was stilloccluded. A plain radiograph of the left scapula showed extensivesclerosis of the tumor, and attempts to do a fine needle aspirationbiopsy of the healed mass were unsuccessful due to its hard, bonynature.

[0076] Detailed Results

[0077] Patient 1. Patient 1 was a 45 year old male. The patient wasdiagnosed to have hepatoma of the right lobe of the liver confirmed byfine needle aspiration biopsy (FNAB). The patient was consideredunsuitable for surgery, radiotherapy and chemotherapy due to poorgeneral condition and large size of the tumor. The patient had lost morethan 25 kgs prior to confirmation of the diagnosis. Prior to thetreatment, a pronounced “tumor blush” was observed in angiograms. In afirst treatment, 1.1 gm of PUFA salt was administered. More than 50% ofthe tumor-feeding vessels were occluded following the first injection,and a significant amount of resistance was noted while injecting thefirst dose of PUFA salt. A repeat angiogram done 4 days after the firstdose of the PUFA salt showed that the tumor blush was much reduced. Atthat time, an additional dose of 0.5 gm of PUFA was administered. Athird angiogram performed 1 week after the second dose of PUFA salt (andthe 11th day after the first dose) showed almost complete occlusion ofthe tumor-feeding vessels. No such occlusion was seen in the normalvasculature feeding non-neoplastic tissues. A >50% reduction in the sizeof the hepatoma in patient 1 was seen one month after the first PUFAsalt injection. The decrease in the size of hepatoma was associated withan increase in the radiographic density of the contrast agent. Thissuggests that, as the size of the tumor was decreasing, there was aconcomitant increase in the density of the contrast agent (to which thePUFA salt was conjugated) in the remaining portion of the tumor. Thepatient felt well for 6 months following the injection, gained >10 kgsin weight, and was asymptomatic. Unfortunately, the patient died in atraffic accident.

[0078] Patient 2. Patient 2 was a 50 year old male. The patient wasdiagnosed to have hepatoma of the right lobe of the liver confirmed byFNAB, and was considered to be high risk for surgery, and unlikely torespond to radiotherapy and/or chemotherapy. During the course ofinjecting the PUFA salt, significant resistance was felt and thereforeonly 750 mg of PUFA salt was administered into the right hepatic artery.An angiogram recorded immediately after the injection showed completeocclusion of the tumor-feeding vessels. The patient experienced a mildto moderate degree of pain in the hepatic region during and after theinjection, which subsided after administering NSAIDs (ibuprofen). Thepain subsided after 24 to 48 hours. This patient developed peritonitis 1week after the PUFA administration. Subsequent investigations revealedthat the patient had developed a perforated duodenal ulcer, which wasresponsible for the peritonitis but presumably unrelated to the PUFAinjection. The patient died on the 10th day due to complications of theperforated duodenal ulcer.

[0079] Patient 3. Patient 3 was a 24 year old male. The patient wasdiagnosed to have giant cell tumor (osteoclastoma) involving the medialcondyle of the right femur. The diagnosis was confirmed by biopsy. Thepatient refused the recommended surgical amputation above the rightknee, and opted to try a PUFA injection. A total dose of 500 mg of PUFAsalt was delivered into the femoral/popliteal artery. Complete occlusionof the tumor-feeding vessels was noted immediately after the injection.The patient complained of pain in the right knee area and was givenNSAIDs. A repeat angiogram done 10 days after the injection of PUFA saltshowed that the occluded tumor-feeding vessels remained closed. Thepatient appeared for a follow-up 2 months after the injection, at whichtime he reported that he was better except for mild pain in the regionof the tumor. The patient was subsequently lost for follow up visits.

[0080] Patient 4. Patient 4 was a 30 year old male. The patient wasdiagnosed to have giant cell tumor of the left scapula, confirmed bybiopsy. The patient refused the recommended surgical removal of the leftupper limb and opted to try a PUFA injection. The patient received 660mg of PUFA salt through selective catheterization of the left subclavianartery. During the course of the injection, significant resistance wasfelt and the injection was stopped after delivering 660 mg of PUFA salt.Complete occlusion of the tumor-feeding vessels was seen immediatelyafter the administration of PUFA salt. This patient was discharged fromthe hospital after 24 hours of observation. The patient felt better andwas able to do use his left arm normally, without any pain or limitationof movement. Seven and a half years after the PUFA salt injection, anangiogram showed that the tumor-feeding vessels were still occluded andthat the tumor mass was completely calcified. Attempts at FNAB were notsuccessful due to hard nature of the healed mass.

[0081] Patient 5. Patient 5 was a 79 year old male. This patient wasdiagnosed to have right renal cell carcinoma. By the time it wasdiagnosed, the disease was well advanced, with secondary tumors in theliver, peritoneum and right pleural effusion. The patient had lost about10 kgs of weight and was considered a high risk for surgery. In a firsttreatment, 750 mg of PUFA salt was delivered into the right renal arteryvia the right femoral route. Complete occlusion of the tumor-feedingarteries was noted without any occlusion of the normal arteries whichwere feeding the normal lower pole of the right kidney. The patientexperienced mild to moderate pain at the site of tumor (right lumbararea). The pain subsided after administering NSAIDs. The patientcomplained of pain in the right knee area and was given NSAIDs (buscopanby injection and oral ibuprofen). The patient was sent home 48 hoursafter PUFA salt injection. The patient survived for 3 months, and thendied due to extensive metastasis in the liver and lungs.

[0082] Equivalents

[0083] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising apolyunsaturated fatty acid, or a salt of a polyunsaturated fatty acid,in combination with a lymphographic agent.
 2. A pharmaceuticalcomposition as in claim 1 wherein said lymphographic agent is an iodizedfatty acid.
 3. A pharmaceutical composition as in claim 1 wherein saidlymphographic agent is combined with said polyunsaturated fatty acid ina solution.
 4. A pharmaceutical composition as in claim 1 wherein saidlymphographic agent is conjugated to said polyunsaturated fatty acid. 5.A pharmaceutical composition as in any one of claims 1-4 wherein saidpolyunsaturated fatty acid is an essential fatty acid.
 6. Apharmaceutical composition as in claim 5 wherein said essential fattyacid is selected from the group consisting of gamma-linolenic acid,arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid,di-homo-gamma-linolenic acid, alpha-linolenic acid, linoleic acid, andconjugated linoleic acid.
 7. A pharmaceutical composition as in any oneof claims 1-4 wherein said polyunsaturated fatty acid is administered inthe form of a salt selected from the group consisting of a lithium salt,a sodium salt, a potassium salt, a magnesium salt, a calcium salt, amanganese salt, an iron salt, a copper salt, an aluminum salt, a zincsalt, a chromium salt, a cobalt salt, a nickel salt and an iodide.
 8. Apharmaceutical composition as in any one of claims 1-4 wherein saidpolyunsaturated fatty acid is in the form of a fatty acid derivativeselected from the group consisting of glycerides, esters, free acids,amides, phospholipids and salts.
 9. A pharmaceutical composition as inany one of claims 1-4 further comprising an anti-neoplastic agent.
 10. Apharmaceutical composition as in claim 9 wherein said anti-neoplasticagent is selected from the group consisting of tumor necrosis factor, ananti-cancer drug, a lymphokine, and specific polyclonal or monoclonalantibodies.
 11. A pharmaceutical composition as in claim 10 wherein saidlymphokine is selected from the group consisting of alpha interferon andgamma interferon.
 12. A pharmaceutical composition as in claim 10wherein said anti-cancer drug is selected from the group consisting ofvincristine, adriamycin, doxorubicin, cyclophosphamide, cis-platinum,L-asparaginase, procarbazine, camptothecin, taxol and busulfan.